Genomic characterization and immunotherapy for microsatellite instability-high in cholangiocarcinoma

Background Microsatellite instability-high (MSI-H) is a unique genomic status in many cancers. However, its role in the genomic features and immunotherapy in cholangiocarcinoma (CCA) is unclear. This study aimed to systematically investigate the genomic characterization and immunotherapy efficacy of MSI-H patients with CCA. Methods We enrolled 887 patients with CCA in this study. Tumor samples were collected for next-generation sequencing. Differences in genomic alterations between the MSI-H and microsatellite stability (MSS) groups were analyzed. We also investigated the survival of PD-1 inhibitor-based immunotherapy between two groups of 139 patients with advanced CCA. Results Differential genetic alterations between the MSI-H and MSS groups included mutations in ARID1A, ACVR2A, TGFBR2, KMT2D, RNF43, and PBRM1 which were enriched in MSI-H groups. Patients with an MSI-H status have a significantly higher tumor mutation burden (TMB) (median 41.7 vs. 3.1 muts/Mb, P < 0.001) and more positive programmed death ligand 1 (PD-L1) expression (37.5% vs. 11.9%, P < 0.001) than those with an MSS status. Among patients receiving PD-1 inhibitor-based therapy, those with MSI-H had a longer median overall survival (OS, hazard ratio (HR) = 0.17, P = 0.001) and progression-free survival (PFS, HR = 0.14, P < 0.001) than patients with MSS. Integrating MSI-H and PD-L1 expression status (combined positive score ≥ 5) could distinguish the efficacy of immunotherapy. Conclusions MSI-H status was associated with a higher TMB value and more positive PD-L1 expression in CCA tumors. Moreover, in patients with advanced CCA who received PD-1 inhibitor-based immunotherapy, MSI-H and positive PD-L1 expression were associated with improved both OS and PFS. Trial registration This study was registered on ClinicalTrials.gov on 07/01/2017 (NCT03892577). Supplementary Information The online version contains supplementary material available at 10.1186/s12916-024-03257-7.


Background
The mismatch repair (MMR) pathway is essential for maintaining DNA replication fidelity, mutation avoidance, and genomic stability [1].When the MMR pathway is not functioning correctly, cells are unable to correct errors during DNA replication, leading to an inconsistent number of microsatellite nucleotide repeats and instability in microsatellite regions [2].This deficiency, known as deficient DNA mismatch repair (dMMR), is characterized by microsatellite instabilityhigh (MSI-H) in tumors and has a unique genomic status and tumor microenvironments [3,4].Patients with MSI-H cancers, including gastrointestinal (GI) cancer, have experienced excellent effects from programmed cell death protein-1 (PD-1)-based immune checkpoint inhibitor (ICI) immunotherapy [5][6][7].Based on this evidence, the Food and Drug Administration (FDA) approved programmed cell death protein-1 (PD-1) inhibitors monotherapy (and cytotoxic T lymphocyte-associated antigen 4 inhibitors) for patients with MSI-H/dMMR colorectal cancer and multiple other cancers, regardless of the location [3,[8][9][10][11].
The impact of MSI-H status on the genomic profile and immunotherapy response in CCA is very important but has not been well characterized.In this study, we systematically analyzed the genomic characteristics and immunotherapy efficacy of MSI-H by comparing microsatellite stability (MSS) status in patients with CCA.Integrating histopathological features to identify potentially beneficiary patients for immunotherapy with a MSS status is also very important.

Patient characteristics
This study involved 887 patients who were diagnosed with histologically confirmed CCA and treated at the Peking Union Medical College Hospital (PUMCH) from 2017 to 2020 and The Affiliated Hospital of Qingdao University (QDUH) from 2017 to 2019.All MSI-H CCA patients were enriched by hand searches of medical records as much as possible to allow better comparability between the two groups.The study collected samples including tumor tissues and paired tumor-free tissues, and clinical data such as gender, age, tumor location, tumor size, tumor differentiation, lymph node metastasis, and tumor stage from electronic medical records.Before sample disposal, two independent pathologists reviewed all tumor tissues to confirm the pathological diagnoses.Only samples with an estimated tumor purity of > 20% on histopathological assessment were subjected to genomic profiling.The study obtained written informed consent from all participants and was approved by the Institutional Ethics Review Committee at PUMCH and QDUH (NCT03892577; RWSHBC).This study followed the guidelines of the Declaration of Helsinki and Good Clinical Practice.

Genomic profiling
Genomic DNA was prepared from formalin-fixed, paraffin-embedded tumor samples and matched white blood cells (84.9%) or paracancerous/normal tissues (15.1%) using a DNA extraction kit (QIAamp DNA FFPE Tissue Kit), according to the manufacturer's protocols.NGS-based cancer sequencing YuanSu (CSYS) panel (n = 824) or WES (n = 63) was performed as previously described in the OrigiMed [27][28][29][30].Tumor samples were sequenced by CSYS panel with a mean coverage of 900 × and matched normal blood samples were sequenced to a mean unique coverage of 300 × .
For WES, the mean coverage was 500 × for the tumor sample and 100 × for the matched normal blood samples on the Illumina NovaSeq6000 Platform (Illumina).
The study determined the functional significance of variants in genes by examining databases and published literature, including ClinVar, Catalogue of Somatic Mutations in Cancer, and PubMed.The study reported known or likely drivers and recurrent variants.Pathogenic mutations were defined as variants that clearly affect the function of a gene [30,31].Synonymous variants and variants with uncertain significance (VUS) (named VUS) were excluded because of lacking clinical significance [32].Only pathogenic, likely pathogenic, and some meaningful mutations that are clear, potentially functionally, clinically significant, or functionally unknown were reported as clear somatic variants (named Not-VUS) in tumors.
The tumor mutation burden (TMB) was calculated by counting the total number of coding mutations per megabase as report [27,33].TMB values higher than 10 muts/Mb were considered TMB-high (TMB-H), and those less than 10 muts/Mb were considered TMB-low (TMB-L) [34].The indel ratio is defined as the number of (frameshift) indel mutations per megabase in the target region [3,35].MSI status was determined based on the MANTIS score [36].MSI-H is defined as more than 15% of selected microsatellite loci showing unstable in tumors compared to matched peripheral blood [29].

PD-1 inhibitor-based immunotherapy cohort of cholangiocarcinoma and efficacy evaluation
In patients who received PD-1 inhibitor-based therapy, follow-up was conducted to evaluate the efficacy and safety of the drugs until overall survival (OS) was determined.Further analysis was performed on patients who meet the following conditions: (1) patients should have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-2 and normal baseline organ functions; (2) patients had at least one measurable lesion used to assess the therapeutic response according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 [38]; (3) patients with radiologically or histologically confirmed advanced CCA who received PD-1 inhibitors based combination therapy with other agents of cancer treatment [39][40][41].
A total of 139 patients with advanced CCA who received PD-1 inhibitor-based combination therapy were enrolled in this study.PD-1 inhibitor-based therapy may have been heterogeneous for different PD-1 inhibitors (including pembrolizumab or nivolumab, toripalimab, camrelizumab, sintilimab, or tislelizumab every three weeks intravenously) and combination therapies in a real-world setting [39][40][41].Patients were followed every six to eight weeks.The treatment effects were evaluated according to the RECIST version 1.1 guidelines [38] by professional radiologists at our center who were blinded to the therapeutic outcomes and clinicopathological features.Durable clinical benefit (DCB) was defined as complete response (CR), partial response (PR), or stable disease (SD) for ≥ 24 weeks; other was defined as no durable benefit (NDB) [42].Progression-free survival (PFS) was defined as the time from initiating anti-PD-1 treatment to the first documented disease progression or death due to any causes, while OS was defined as the time between the start of anti-PD-1 treatment and death due to any causes.

Identification and classification of potentially actionable alterations of cholangiocarcinoma
The OncoKB database was used to determine potentially actionable targets (PATs) of genetic alterations in druggable target [43].All actionable alterations were classified as levels 1, 2, 3A/B, and 4 as follows.Level 1 means FDA-recognized biomarker predictive of response to an FDA-approved drug in this indication; while Level 2 means standard care biomarker recommended by the National Comprehensive Cancer Network (NCCN) or other professional guidelines predictive of response to an FDA-approved drug in this indication.Level 3A means compelling clinical evidence supports the biomarker as being predictive of response to a drug in this indication; while Level 3B means standard care or investigational biomarker predictive of response to an FDA-approved or investigational drug in another indication.For Level 4, this means there is compelling biological evidence to support that the biomarker can predict drug response [43].Usually, the alterations of levels 1, 2, and 3A were deemed actionable targets [28,30].

Statistical analysis
The study assessed normal distribution was assessed using the Kolmogorov-Smirnov test.Fisher's exact test was utilized to compare categorical variables between multiple groups.For continuous variable comparisons between two groups, a two-tailed unpaired t-test was used when the data were normally distributed; otherwise, Wilcoxon's test was used.The study identified variables associated with MSI-H using logistic regression analysis.For survival analysis, univariable and backward stepwise multivariable Cox proportional hazards regression models (if univariate P value of < 0.1) were used to calculate hazard ratios (HRs).Kaplan-Meier plots (log-rank tests) were used to describe the prognostic factors related to PFS and OS.The results are reported as HRs and 95% confidence intervals (CIs).The R package "survival" for survival analysis, "forestplot" for forest maps, and "ComplexHeatmap" for profiling heat maps were used.The function "soma-ticInteractions" from R package "maftools" was used to detect mutually exclusive or co-occurring mutations in MSI-H and MSS groups [49].All reported P values were two-tailed, and P < 0.05 was considered statistically significant.All statistical analyses were performed using R version 4.1.1.

Clinical characteristics of patients with CCA
A total of 887 patients with CCA were included in this study; 584 (65.8%) had intrahepatic cholangiocarcinoma (ICC) and 303 (34.2%) had extrahepatic cholangiocarcinoma (ECC).The median patient age was 60 years old (range: 19-89 years old).Among these patients, 60.2% were male, and 5.4% (48/887) were identified as MSI-H.The median TMB value was 3.7 (interquartile range [IQR]: 1.9-6.2) muts/Mb.The clinicopathological characteristics of the patients and individual details are shown in Additional File 2: Table S2 and Additional File 1: Table S3.

Univariate and multivariate survival analyses of the CCA cohort receiving PD-1 inhibitor-based therapy
Due to the small number of MSI-H patients, potential benefits from histopathological and clinical factors should be identified to confirm the efficacy of immunotherapy for patients with advanced CCA.

Potentially actionable targets (PATs) and co-altered mutations in CCA with different MSI statuses
We used the OncoKB database to annotate the PATs and found that all the MSI-H groups had level I evidence of pembrolizumab use; TMB-H was also considered to have level I evidence of pembrolizumab use.So, only 8.7% of patients in the MSS group (with TMB-H) fulfilled level I evidence for the use of pembrolizumab (Fig. 5A and B).Except for the targets of MSI-H and TMB-H, only 2.08% of patients had a PAT gene mutation (≤ level 3A) in the MSI-H group, compared with 10.25% in the MSS group (Fig. 5C and D).In the MSS group, IDH1, KRAS, BRAF mutation, and FGFR2 fusion were the main target genes (Fig. 5E and F).We further explored the underlying differences of co-altered mutations in MSI-H and MSS groups.Cooccurrence mutation statuses in the MSI-H group were observed between ARID1A:NOTCH3, ARID1A:KRAS, KMT2D:LRP1B, MSH3:OBSCN, and ATM:POLE.However, NF1:LRP1B was identified exclusively.On the other hand, in the MSS group, TP53:CDKN2A, TP53:TERT, TP53:FBXW7, KRAS:SF3B1, and KRAS:CDKN2A were identified co-occurrence; however, TP53 mutants with STK11, ATM, IDH1, or BAP1 mutations; and KRAS mutation with ERBB2, BRAF, NF1, IDH1, BAP1, or TERT mutations were identified exclusively (Additional File 2: Figure S6).

Discussion
Our study comprehensively investigated the genomic alterations and PATs for precision medicine according to MSI status in 887 patients with CCA.Tumors with MSI-H status were associated with a higher TMB value and more positive PD-L1 expression but fewer PATs.Furthermore, by investigating immunotherapy in 139 advanced CCA patients, we found that MSI-H patients had longer PFS and OS than MSS patients receiving PD-1 inhibitor-based therapy.By integrating MSI-H with positive PD-L1 expression, we identified certain patients with advanced CCA who may benefit from PD-1 inhibitorbased immunotherapy.
MSI-H cancers have distinct genomic features, and this study chose non-VUS gene alterations for further analysis to emphasize clinical translational precision medicine.The mutation frequencies of ARID1A, ACVR2A, TGFBR2, KMT2D, and RNF43 were significantly higher in the MSI-H group of our cohort.We found that the TMB value of the MSI-H group was higher than those of the MSS group.Abnormal pathways, including the DDR, SWI/SNF, and HRD, were highly frequent (> 80%) in the MSI-H group.Similarly, Eluri et al. reported that the frequency of MSI-H in 7565 ICC cases was 1.8%, and the median TMB of MSI-H patients was 21.7 muts/Mb.Meanwhile, the study also showed that genomic alterations in TP53 (59.9%),PRBM1 (37.2%),ARID1A (13.9%), and APC (13.9%) were enriched in patients with MSI-H [50].In another study, RNF43 and KMT2D mutations frequently occurred in CCA patients with MSI-H and TMB-H status [51].Similarly, an analysis of the genomic landscape of MSI-H in 11,395 tumors across 30 cancer types showed that mutations in ACVR2A (73%), KMT2D (68%), KMT2B (66%), and MMR-related genes were enriched in the MSI-H group [52].
Tumors with MSI-H have dysfunctions that correct errors introduced in microsatellites, which could lead to more frameshift mutations, higher TMB value, and increased neoantigens formation and tumor-specific antigens which are thought to be unknown and novel to the individual immune system [53].Then, tumors with MSI-H are highly infiltrated with cytotoxic T-cell lymphocytes like CD8 + T-cells with a Tc1 phenotype and CD4 + T-cells with a Th1 phenotype, to raise tumorspecific immune responses, which leads to sensitivity to ICI of MSI-H tumors [54].Some studies have found that MSI-H in patients with CCA has promising efficacy in immunotherapy, however, the patients' numbers were small [21, 23-26, 50, 55, 56].In our cohort with 139 patients, we found that CCA patients with MSI-H had longer median OS (HR = 0.17, P = 0.001) and PFS (HR = 0.14, P < 0.001) than CCA patients with MSS, indicating that patients with MSI-H may have more opportunities to benefit from immunotherapy.In the KEYNOTE-158 study, 22 advanced CCA patients with MSI-H/dMMR received pembrolizumab treatment and ORR could reach up to 40.9%, with median PFS and median OS of 4.2 (2.1-24.9)and 19.4 (6.5-NR), respectively; ≥ 3 years OS rate exceeded 30% [23].However, for the 104 advanced BTC patients with MSS status that received pembrolizumab treatment, the ORR was only 5.8%, with median PFS and median OS of only 2.0 (1.9-2.1) and 7.4 (5.5-9.6)months, respectively; the survival rate of ≥ 3 years was only about 10% [57].These results suggested that patients with MSI-H have more opportunities to benefit from immunotherapy.In our study, MSI-H status was a protective factor for both OS and PFS of immunotherapy in multivariate analysis.However, another study found that MSI-H (N = 15) was not an independent protective factor (P = 0.162) in refractory CCA with PD-L1 positivity (CPS≥ 1 or tumor proportion score (TPS) ≥ 1%) and treated with pembrolizumab, indicating that PD-L1 expression is also another important confounding factor [58].
Our study found that positive PD-L1 expression was relatively high in patients with MSI-H status.In many types of GI cancers, including BTC, colorectal cancer, and gastric cancer, PD-L1 expression was significantly associated with MSI-H status (P < 0.001) [22,59,60].In our study, we observed that the combination of MSI status and PD-L1 expression could distinguish the prognosis of immunotherapy for both OS and PFS.We found patients with PD-L1 CPS ≥ 5 had a better both OS and PFS than patients with CPS < 5 in multivariate analysis, which is consistent with our previous study [61][62][63][64].However, the significance of PD-L1 expression as a biomarker for BTC immunotherapy remains controversial.Yoon et al. reported that a PD-L1 CPS ≥ 1 does not distinguish well from the immunotherapy response in BTC [45].Another study showed that the median PFS and OS of BTC patients did not differ according to the CPS cutoff values (1, 5, and 10) (P > 0.05 for all) [47].Zhou et al. found a correlation between PD-L1 expression in patients with CPS ≥ 5 and CPS < 5 and the efficacy of anlotinib along with the PD-1 inhibitor; but they observed a similar median PFS (6.80 vs. 6.24 months, P > 0.05) [62].In another prospective trial, the high PD-L1 expression (CPS ≥ 5) group did not show any differences in median PFS compared with the low PD-L1 expression group (high vs. low: 5.0 vs. 6.4 months, p = 0.81) [65].Therefore, although patients with PD-L1 CPS ≥ 5 may benefit from PD-1 inhibitor immunotherapy for both OS and PFS in our cohort, the significance of PD-L1 expression and cutoff values still need to be confirmed in prospective and large retrospective cohorts.
We found that the TMB value of the MSI-H group was significantly higher than that of the MSS group, which is a consensus and consistent with the results of other studies involving many cancers [66].In addition, we also found more indel alterations in the MSI-H CCA group, similar to those observed in colorectal cancer [67].Meanwhile, our results also showed that mutated pathways, such as SWI/SNF, BER, MMR, and HRR, mostly contributed to the MSI-H status.Many studies have found a high frequency of MSI-H mutations in the SWI/SNF pathway, including intestinal and pancreatic cancers [68][69][70].Wang et al. defined co-mutations (combinations of HRR-BER and HRR-MMR) in the DDR pathway and found that co-mutated DDR was significantly associated with MSI-H and TMB-H status [37].Mutations in the MMR pathway surely contribute to the MSI-H status [53].Similarly, Zhou et al. found that the percentage of MSI-H patients in the HRR-mut group was higher than that in the HRR-WT group in patients with colon cancer [71].However, in the multivariate Cox analysis of immunotherapy efficacy in our cohort, the prognostic values of TMB, SWI/SNF pathway, or MMR pathway mutations were not significant (P > 0.05).
The percentage of altered genes that can be used as therapeutic targets (level ≤ 3A) according to the OncoKB database in the MSI-H group is low, while the percentage of altered genes in MSS group for potential therapeutic targets could be up to 10%, mainly including IDH1 (ivosidenib), KRAS (adagrasib), FGFR2 fusion (infigratinib), and BRAF mutation (dabrafenib plus trametinib).It showed the spectrum of precision medicine is different between the two groups.Nearly 8.7% of the patients belonged to the TMB-H group in the MSS group, while all (100%) of the MSI-H patients belonged to the TMB-H group, which can also be recommended to use the PD-1 inhibitor pembrolizumab (level I).Similarly, Eluri et al. also observed that MSI-H cases had lower frequencies of previously identified, actionable ICC drivers, such as FGFR2 fusions (0% vs. 9%, p = NS), IDH1 (3.7% vs. 14.5%, p < 0.0001), and IDH2 (0% vs. 4.1%, p = 0.007) [50].Different gene alterations and co-altered patterns may partly explain the low prevalence of PAT for other precision medicines except for ICI in the MSI-H CCA group [51].MSI-H patients with CCA seem only amenable to ICIbased therapy.
Our study provided a large cohort and a great resource to comprehensively investigate genomic alterations and immunotherapy in patients with CCA.However, this study has some limitations.First, from a retrospective perspective, the "enrichment" process of patients with MSI-H tumors may have overestimated the proportion of the natural prevalence of MSI-H.However, this study focused on assessing genomic features and comparing immunotherapy efficacy between the two groups, so we wanted to enroll as many MSI-H patients as possible.Second, we just compared DNAlevel characteristics according to MSI status, we hope to analyze the multi-omics feature (including RNA sequencing and immune marker by IHC) between MSI-H and MSS groups further.Third, in our immunotherapy cohort, the heterogeneity of PD-1 inhibitor-based therapy may stem from variations across different PD-1 inhibitors and combination therapies.But we found MSI status and PD-L1 CPS ≥ 5 could stratify the cohort prognosis in log-rank tests and multivariable Cox analysis.We encourage validating our findings in other large cohorts.

Conclusions
The proportion of MSI-H in CCA was low.The mutation spectrum was different between MSI-H and MSS status.The MSI-H status of patients with CCA was associated with TMB-H and positive PD-L1 expression.Moreover, MSI-H and positive PD-L1 expression were associated with improved both OS and PFS in patients with advanced CCA who received PD-1 inhibitor-based immunotherapy.

Abbreviations
We analyzed the association between mutations of 15 pathways and important clinicopathological parameters (like age, gender, tumor location), and MSI-H status, to analyze what factors contribute to MSI-H status.Results showed that the SWI/SNF (OR = 38.07),BER (OR = 19.14),MMR (OR = 10.23),HRR (OR = 9.68), PI3K (OR = 6.37), and WNT (OR = 4.65) pathways have contributed to MSI-H status by multivariate logistic

Fig. 4 Fig. 5 Fig. 5 (
Fig.4 Factors contributing to MSI-H status.Using multivariate logistic regression, the association between pathways and clinical factors, and MSI-H status were analyzed.MSI-H: microsatellite instability-high